A system for the construction of targeted unmarked gene deletions in the genus Burkholderia

Abstract

Burkholderia species are extremely multidrug resistant, environmental bacteria with extraordinary bioremediation and biocontrol properties. At the same time, these bacteria cause serious opportunistic infections in vulnerable patient populations while some species can potentially be used as bioweapons. The complete DNA sequence of more than 10 Burkholderia genomes provides an opportunity to apply functional genomics to a collection of widely adaptable environmental bacteria thriving in diverse niches and establishing both symbiotic and pathogenic associations with many different organisms. However, extreme multidrug resistance hampers genetic manipulations in Burkholderia. We have developed and evaluated a mutagenesis system based on the homing endonuclease I-SceI to construct targeted, non-polar unmarked gene deletions in Burkholderia. Using the cystic fibrosis pathogen Burkholderia cenocepacia K56-2 as a model strain, we demonstrate this system allows for clean deletions of one or more genes within an operon and also the introduction of multiple deletions in the same strain. We anticipate this tool will have widespread environmental and biomedical applications, facilitating functional genomic studies and construction of safe strains for bioremediation and biocontrol, as well as clinical applications such as live vaccines for Burkholderia and other Gram-negative bacterial species.

Relating the Physical Properties of Pseudomonas aeruginosa Lipopolysaccharides to Virulence by Atomic Force Microscopy

“…monocytogenes (19). To determine if the measured adhesion forces could serve as a predictor of virulence for the P. aeruginosa Wzz mutants, we tested each of the deletion mutants in an acute pneumonia model of infection using BALB/c mice.…”

“…P. aeruginosa containing the resolution vector was selected for on L agar plates with spectinomycin at 100 g/ml and tetracycline at 100 g/ml, and colonies were screened for resolution of the chromosomal pGPI vector via PCR. Maintenance on L agar without antibiotic resulted in the loss of pDAI (19). The resulting deletion derivatives were referred to as ⌬Wzz1 and ⌬Wzz2.…”

“…Each deletion mutant construct was excised from Topo2.1 using the EcoRI sites and ligated into the EcoRI-cut pGPI vector, creating the pGPI-⌬wzz1 and pGPI-⌬wzz2 vectors. Ligations were precipitated and electroporated directly into Escherichia coli strain Sy327 (19).…”

BcsKC Is an Essential Protein for the Type VI Secretion System Activity in Burkholderia cenocepacia That Forms an Outer Membrane Complex with BcsLB

“…Plasmids-Unmarked and nonpolar deletions were performed as described previously (39). Details about the construction of the deletion plasmids are available in the supplemental materials.…”

Two type IV secretion systems with different functions in Burkholderia cenocepacia K56-2

“…Because sacB gene selection to screen for double-crossover transconjugants does not function in B. cenocepacia (Barrett et al, 2008;Flannagan et al, 2008), only Tp r Tc s transconjugants were considered to be putative doublecrossover mutants. The target gene deletion was confirmed by using multiple PCR set combinations, both internal and external to the target deletion, and by Southern blot analysis using the Tp cassette and PCR products of sequence immediately downstream of bcvirD4 as probes (data not shown).…”